Computational protocol: Pyrazinamide Susceptibility and pncA Mutation Profiles of Mycobacterium tuberculosis among Multidrug-Resistant Tuberculosis Patients in Bangladesh

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Protocol publication

[…] We sequenced the pncA gene of all isolates. The sequencing was done according to procedures described earlier (). Briefly, the pncA gene was amplified by PCR using forward primer 5′-GGTCATGTTCGCGATCGTCG-3′ and reverse primer 5′-ACAGTTCATCCCGGTTCGGC-3′. Each 25-μl PCR mixture contained 12.5 μl HotStar Taq master mix (Qiagen Inc., Valencia, CA, USA), 0.15 μl each of 50 μM forward and reverse primers, 7.2 μl of nuclease-free water, and 5 μl of genomic DNA. PCR was performed on a MyCycler instrument (Bio-Rad, Hercules, CA, USA) with an initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s and a final extension at 72°C for 7 min. PCR products were analyzed on 2% agarose gels and were purified using a MinElute PCR purification kit (Qiagen Inc., Valencia, CA, USA). The purified PCR products were sequenced at ICDDR,B using an ABI 3500xL genetic analyzer (Applied Biosystems). The resulting raw sequences were analyzed by Chromas software (version 2.33) and aligned with the WT sequence using ClustalW multiple-sequence alignment incorporated into BioEdit sequence alignment editor software (version 7.2.6). […]

Pipeline specifications

Software tools Chromas, Clustal W, BioEdit
Application Sanger sequencing
Organisms Homo sapiens, Mycobacterium
Diseases Tuberculosis, Tuberculosis, Meningeal, Tuberculosis, Multidrug-Resistant
Chemicals Pyrazinamide